Summary: Preclinical studies and clinical trials with agents such as anti-CD3 mAb, LFA3Ig, anti-thymocyte globulin, CTLA4Ig, and cyclosporin A have shown that Type 1 diabetes (T1D) is caused by T cell killing of ? cells, most likely mediated by CD8+ T cells. In our previous clinical trials of anti-CD3 mAb (teplizumab) supported by this grant and mechanisms that we studied in trial participants and others with T1D, we showed that a single course of mAb attenuated the loss of C-peptide compared to control subjects over the first 2 years of disease, even in patients who had T1D for more than 4 months. We also showed that CD8+ T cells were the cells most affected by the drug and changes in these cells accounted for the clinical efficacy. There is, however, limited information about how immunologics change the antigen specific CD8+ T cell responses that are thought to cause the disease. The overall goal of this renewal proposal is to build on our previous clinical and laboratory based studies to identify the role of CD8+ T cells in T1D and determine whether and how these cells are affected by immune therapy. We have developed novel methods for identifying antigen specific CD8+ T cells in HLA-A2+ subjects and tracking them over time in vivo based on creation of CD8+ T cell libraries. Also, under this funding mechanism, we developed a novel assay to measure ? cell death in vivo. Our preliminary data shows that in individuals with T1D, there is an increased frequency of autoantigen reactive memory CD8+ T cells compared to healthy control subjects. The antigen reactive cells are generally reactive to a single diabetes peptide ? most commonly ZnT8186-194, whereas non-diabetic and even non-diabetic identical twins of a patient respond to other peptides. We will test the hypothesis that in those with diabetes and at-risk relatives CD8+ T cells reactive to many antigens can be found but in those who progress to T1D, there is focusing of the response to a limited repertoire of autoantigens. We have identified unique patient groups whose samples will enable us to address this hypothesis: discordant and concordant twins and triplets and at-risk relatives of patients. We will test the frequency, function, and phenotype of the antigen reactive T cells. Using autologous iPS derived ?-like cells derived from patients, we will test whether autoantigen specific CD8+ T cells kill ? cells. We will use clinical samples from the anti-CD3 mAb prevention trial to determine whether anti-CD3 mAb deletes autoreactive CD8+ T cells or renders them exhausted or anergic, and the relationship between the changes in these cells, clinical responses, and ? cell death. Finally, T1D has been shown to be more aggressive in children than adults yet children show a more robust response to most immune therapies. We will address this critical question using cellular studies of the autoantigen reactive cells and their responses to teplizumab. The results of our studies will not only develop an understand the role of autoantigenic CD8+ T cells in T1D but will also provide guidance to the use of immune therapies or antigen targeted therapies to prevent or treat the disease.